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The cytoplasmic areas of integrins, which are important mediators of cell adhesion, have a role in the regulation of cell shape, growth, motility and differentiation. The development of calreticulin-deficient embryonic stem cells and isolated embryonic fibroblasts from calreticulin mutant mice is described. Integrin-mediated adhesion was found to be severely impaired and cytosolic calcium was absent from calreticulin-deficient cells. This indicates that calreticulin is essential in the modulation of both integrin adhesive functions and integrin signalling.

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence life-time imaging, has been used to estimate the size of oligomers formed by the M2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M2 receptor to enhanced green fluorescent protein-M2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values ore as follows: n = 2, 0.495 ± 0.019; n = 4, 0.202 ± 0.010; n = 6, 0.128 ± 0.006; n = 8, 0.093 ± 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20 - 0.24, identifying the M2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process. [ABSTRACT FROM AUTHOR]